This invention relates to polymerizable macromers containing carbohydrates including with N-Acetyl Glucosamine (NAG) of molecular weight ranging between 700 Daltons to 1,00,000 Daltons having formula herein below. 
wherein,
R is H, CH3, C2H5, C6H5,
R1 is H, CH3, C2H5, C6H5 
X may be between 4 to 10, n is from 2 to 50
Y may be N-Acetyl Glucosamine (NAG), mannose, galactose, sialic acid, fructose, ribulose, erythrolose, xylulose, psicose, sorbose, tagatose, glucopyranose, fructofuranose, deoxyribose, galactosamine, sucrose, lactose, isomaltose, maltose, cellobiose, cellulose and amylose.
More particularly it relates to the said polymerizable macromers containing various carbohydrate ligands including with NAG and preparation thereof through the specific linkage mentioned herein. Still more particularly it relates to macromer, which bind more strongly to lysozyme than NAG itself.
The macromers of the present invention as mentioned above are prepared by coupling acryloyl-spacer conjugate of formula (2) claimed in our copending Patent Application no NF363/02 entitled xe2x80x9cOligomer and preparation thereofxe2x80x9d herein below 
wherein,
R is H, CH3, C2H5, C6H5 X may be between 4 to 10.
with functional polyvalent oligomers comprising NAG, sialic acid, galactose or mannose exemplified with NAG as herein given below having Formula (3) 
wherein, n=2 to 50
The polymerizable macromers may be used for inhibition of viral infections and the recoveries of biomolecules. The approach of synthesis of polymerizable macromers with ligand N-Acetyl Glucosamine (NAG) is a generic and can be used for other ligands such as sialic acid, galactose and mannose.
Carbohydrates play a crucial role in biological phenomena and therefore these molecules have attracted the attention of chemists and biochemists. These biomolecules are ubiquitous, figuring prominently in various processes such as cell differentiation, cell growth, inflammation, viral and bacterial infection, tumorigenesis and metastasis (Rouhi A., M., C and EN, Sep. 23,62-66,1996).
Many infections caused by bacteria and virus are a result of host receptor interactions. The foremost step for the infection is the adhesion of the ligands present on the infectious microbe to the receptors of the host cells. Adhesion and interactions have to be strong for a successful infection. If the adhesion is not adequate then normal defense mechanism can intercept this process. Viruses and bacteria for example interact with certain saccharides of the host cell. Bacteria express a large number of lectins and are used to adhere to glycocalyx of the host cell through a multivalent interactions. Agglutination of erythrocytes is a case in point.
Carbohydrates exhibit molecular diversity and wide structural variations, which makes carbohydrates alternative ligands for competitive binding to inhibit the infections.
Many alterations and modifications of the naturally occurring O-/N-glycosidic sugars are being reported and is an area of prime interest to the chemist and biochemist.
The importance of carbohydrates in biologically relevant recognition processes has been recognized fairly recently (Feizi, T., Biochem. J. 245:1,1987). In addition, carbohydrates on cell surfaces play an important role in intercellular communication and recognition processes, which is principally based on receptor-ligand interactions.
Carbohydrates are usually attached to other moieties such as lipids or proteins. Belvilacqua et al., (Science, 243:1160,1989) have demonstrated the role of carbohydrates along with proteins and nucleic acids as a primary biological information carriers.
The inventors of the present invention have observed that it may be worthwhile to use carbohydrates in therapeutics for human, especially since they can play an important role in prevention of viral and bacterial infections. Recently few reports have been published to justify the use of carbohydrates. Krepinsky et al. (U.S. Pat. No. 6,184,368, 2001) suggested the application of carbohydrates in preventing the infections. Mandeville, et al. (U.S. Pat. No. 5,891,862,1999) reported the use of polyvalent polymers containing carbohydrates for the treatment of rotavirus infection
Polyvalent molecules bind to the receptor molecules through multiple contacts, which results in strong binding. However the synthesis of ligands is critical and involves multiple steps. The polyvalent interactions can be maximized by incorporation of ligands optimally tailored based on the understanding of the binding between the ligand and the host receptor. The enhanced interactions are important especially when the ligands are expensive e.g. sialic acid.
The inventors of the present invention have also observed that interactions of ligand with a receptor can be enhanced by 1) appropriate incorporation of the ligand 2) incorporation of spacer chain and 3) by steric stabilization/exclusion.
Spaltenstein et al., (J.Am.Chem.Soc.,113:686,1991) reported increased interaction between the receptor and ligand due to plurality of binding ligands and the receptors on the host surface. This was illustrated by the influenza virus hemagglutinin, which binds to neuraminic acid on the cell surface, which has a greater affinity for its receptor when a polyvalent structure is presented.
The early phase of infection by viral, parasitic, mycoplasmal and bacterial pathogens, is achieved by specific adhesion to cell surface carbohydrate epitopes (Dimick,et al. (J.Am.Chem.Society, 121,10286-10296,1999). Dwek, et al. (Chem. Rev., 96,693, 1996) reported the initiation of a wide range of human disease is mediated by protein-carbohydrate recognition step.
If relative density and spatial arrangement of ligands incorporated is optimized, then the binding can be substantially enhanced. The enhanced interaction between molecular conjugate with a specific binding site of biomolecule also finds applications in affinity separations, drug delivery and biotechnology.
To imitate and exploit this mechanism there is a need to devise a simple synthetic methodology, which will enhance substrate ligand interactions.
Design of high affinity protein carbohydrate binding systems can provide an alternative strategy for the treatment of infectious diseases e.g. influenza and rotavirus. This has the advantage as such agents will not have pathogen resistance to antibiotics and drugs. A new approach to treat influenza is based on the principle of inhibition of virus to the host cells. The inhibitors like sialic acid anchored to polymeric or liposomal carriers have been reported in the past.
Since monovalent interactions of natural oligosaccharides are weak, they need to be used in large quantities for an effective treatment. This problem can be overcome by synthesizing polyvalent carbohydrate molecules (Zopf, D., Roth, S. Lancet 347, 1017, 1996). The concept is attractive since it would provide a non-toxic therapeutic to a wide range of human diseases. But synthesis of such compounds is critical and requires knowledge of the host-cell binding mechanism.
Polymeric ligands that bind to the virus more powerfully than the Red Blood Cells will prevent the influenza infection. Similar binding is also involved in rotavirus infections. (Mandeville et al. U.S. Pat. No. 6,187,762, 2001)
Advantage of carbohydrate modification lies in that it may impart change in physical characteristics such as solubility, stability, activity, antibody recognition and susceptibility to enzyme. Sharon, et al., (Science,246:227-234,1989) reported carbohydrate portions of glyco-conjugate molecules to be important entity in carbohydrate biology.
Haemagglutination can be prevented by saccharides multivalent glycoconjugates, which bind to the bacterial lectins and thus inhibit bacterial adhesion. (Sigal, et al.,J.Am.Chem.Soc.118:16,3789-3800,1996).
Damschroder, et al. (U.S. Pat. No. 2,548,520,1951) reported high molecular weight preformed polymers conjugated with unsaturated monomers or proteins. Synthesis of high molecular weight materials of this kind generally requires temperatures up to 100xc2x0 C. Such high temperatures are not well tolerated by most of the proteins as they are thermolabile. Thus the methods described are unsuitable for producing polymers of biologically active molecules.
Carbohydrates can be used as functionalized ligands by incorporation into polymer or macromer backbone. The macromer containing polyvalent ligand can be homopolymerized or copolymerized with suitable monomer to form a multivalent conjugate. Multivalent ligand may include shorter oligomers having pendant functional groups that may impart desirable properties to the polymer.
The present invention involves coupling of oligomers comprising NAG and bearing terminal functional groups adequately described and covered in our copending patent application no. NF 363/02 entitled xe2x80x9cOligomer and preparation thereofxe2x80x9d with polymerizable monomers containing vinyl unsaturation optionally containing a spacer to yield a macromers. We have demonstrated that macromers bind to lysozyme more strongly as evidenced by values of Kb and inhibit lysozyme more efficiently as evidenced by values of I 50. 
Multivalent macromers of varied length and density will be useful for receptor ligand interactions of biological origin. Various chemical and chemoenzymatic methods have been reported in the past for the preparation of di and trivalent ligands, dendrimers, and high molecular weight polymers but have proven to be complicated to synthesize.
Thus, there is necessity of a simple methodology to obtain multivalent ligands and polymers of varying chain length.
Mammen, M., and Whitesides, G., M., demonstrated that (J.Med.Chem.38:21,4179-90,1995) agglutination of erythrocytes caused by influenza virus could be prevented by use of polyvalent sialic acid inhibitor. Moreover, they suggested two favorable mechanisms for inhibition between the surfaces of virus and erythrocytes 1) high-affinity binding through polyvalency, and 2) steric stabilization. This novel approach is a model for pathogen-host interactions.
Sigal, et al. (J.Am.Chem.Soc.118:16,3789-3800,1996) prepared polymers containing sialoside and evaluated the efficiency of inhibition of influenza virus in terms of inhibition constants (Ki). Although the authors observed that the extent of inhibition and minimum inhibition concentration decreased with increase in polymer molecular weight and sialic acid content, it was also noted that not all sialic acid ligands were involved in binding with the virus. This clearly indicates need of tailoring the polymer structure so that higher fraction of ligands is involved in binding.
Spevak et al. (J. Am. Chem. Soc., 115,1146-1147, 1993) reported the polymerized liposomes containing C-glycosides of sialic acid, which were potent inhibitors of influenza virus. Moreover the authors demonstrated that the infection was inhibited more effectively when the ligand bearing monomer was polymerized.
Various methods have been reported in the past to synthesize multivalent ligands such as Ring-Opening Metathesis Polymerization (ROMP). ROMP has been used to generate defined, biologically active polymers by Gibson et al., (Chem. Comm., 1095-1096,1997) and Biagini et al., (Polymer, 39, 1007-1014 ,1998).
Carbohydrate receptors also have a role in intracellular trafficking of macromolecules. Therefore, macromolecules containing suitable ligands find applications in biomedical field for e.g. in targeting of drugs to certain tissues and cells in the organisms.
Recent advancements in the field of glycoscience have demonstrated enhanced binding between carbohydrate ligands and specific receptors as a result of the cluster effect. These interactions are result from intrinsic properties of such ligands. Various methods have been reported in the past for the synthesis of glycoconjugate oligomers and the clusters for the receptor binding activity. Nishimora, et al. (Macromolecules, 27, 4876-4880,1994) synthesized sugar homopolymer clusters from acrylamidoalkyl glycosides of N-Acetyl-D-Glucosamine. On addition of the polymer clusters, binding to WGA was enhanced.
The polyvalent interactions have several advantages over monovalent interactions as a result of mode of receptor binding. Moreover, multivalent interactions lead to conformational contact with biological receptors, which subsequently results in enhanced interaction with the substrate.
Previous methods of synthesis of polyvalent ligands are complicated and need higher inhibition concentrations. It is reported that the polymeric fucosides are resistant to neuraminidase enzyme present on the surface of influenza virus. The viruses also cleave sialic acid groups from molecules that bind to the surface of the virus, and thereby destroy the binding ability.
The polymerizable macromers reported by the inventors of the present invention are effective at very low concentration which is a significant advantage when the ligands under consideration are expensive e.g. sialic acid. Further, these macromers can be copolymerized with other comonomers to provide copolymers containing polyvalent ligands. Moreover, the process reported here for the incorporation of polyvalent ligands into polymerizable macromers is relatively simple and involves lesser steps.
The polymerizable macromers are of suitable molecular weights, which can efficiently bind to the target site.
The ligands on the polymerizable macromers have ability to bind to various substrate molecules simultaneously. It is expected that the presence of multiple ligands in the backbone can enhance binding to the viruses and biomolecules. Thus the polymerizable macromer containing multiple ligands at low concentration are utilized and can potentially interact with multiple receptors thereby enhancing the inhibition.
The object of the present invention therefore is to prepare polymerizable macromers comprising polyvalent NAG, which exhibit multivalent interactions and simple and novel process for the preparation thereof. The merits of the approach have been highlighted using NAG as an illustration.
Another object is to provide polymerizable macromers which are more effective in binding with the lysozyme as evidenced by the values of the binding constants Kb and relative inhibition of lysozyme more effectively as evaluated by the values of I 50.
Yet another object is to provide polymerizable macromers for applications in medicine and biotechnology.
Yet another object is to provide a convenient process of preparation of polyvalent ligand NAG, mannose, galactose or sialic acid, fructose, ribulose, erythrolose, xylulose, psicose, sorbose, tagatose, glucopyranose, fructofuranose, deoxyribose, galactosamine, sucrose, lactose,. isomaltose, maltose, cellobiose, cellulose and amylose.
Another object is to provide a convenient process of preparation of polymerizable macromers, in the form of monomers containing Acryloyl, Methacryloyl or Para Vinyl Benzoyl (PVB) moieties.
Yet another object is to provide a convenient process of incorporation of spacer arm to a polymerizable monomer.
Yet another object is to provide a convenient process of conjugation of polymerizable monomer containing a spacer arm and polyvalent ligand.
Yet another object is to provide a process of preparation of polymerizable macromers containing NAG ligands for enhanced interactions.
Still another object is to provide more stable ligands for the interactions with biomolecules than the natural polymers such as chitin and chitosan containing natural ligand NAG.
Therefore, the objective of the present work is to provide polymerizable macromers containing polyvalent ligand for enhanced interactions with the substrates and the process for the preparation thereof.
Chitosan is a linear, binary heteropolysaccharide and consists of 2-aceta amido-2-deoxy-xcex2D-glucose (GlcNAc; A-unit) and 2-amino 2-deoxy-xcex2-D-glucose (GIcNAc, D-unit). The active site of lysozyme comprises sub-sites designated A-F. Specific binding of chitosan sequences to lysozyme begins with binding of the NAG units in the subsite C. Moreover, there is a need to synthesize ligands similar to repeat units of chitosan which will not be hydrolyzed by lysozyme. Moreover natural ligands derived from glucose are susceptible to microbial growth. The polymerizable macromers reported here are stable than chitin and chitosan reported earlier.
In our copending application No. NF 363/02 entitled xe2x80x9cOligomer and preparation thereofxe2x80x9d we have shown that the oligomers of NAG in which the NAG groups are juxtaposed to one another, bind more effectively to lysozyme as reflected in values of binding constant (Kb) and the inhibition concentrations I50.
The present invention provides polymerizable macromers containing NAG for a biomolecular target and method for preparation thereof.
The macromers reported here can be homopolymerized or copolymerized with suitable monomers. The approach described to prepare polyvalent carbohydrate macromer containing NAG ligands is simple and can be used to synthesize other macromeric ligands such as sialic acid which bind to influenza virus and rotavirus. Such macromeric ligands may be even used as antiinfective agents both for prevention and treatment of diseases. Moreover, macromers containing NAG can be anchored to thermoprecipitating polymers that can be used for the recovery of biomolecules such as lysozyme and lectins.
The polymerizable polyvalent macromers provided by the present invention can be used for application in recoveries of biomolecules.
The macromers comprising monomer conjugated with polyvalent ligands may also further be used in the treatment of bacterial or viral infections, and are expected not to cause drug resistance.
The approach described herein is a generic one and can be extended to other systems as well for example sialic acid.
The present invention provides process for the preparation of polymerizable macromers containing polyvalent N-Acetyl Glucosamine. The macromers contain polymerizable monomer conjugated to spacer arm covalently bonded to the polyvalent ligand. The macromers reported in this invention provide improved binding and inhibition even at low concentration. Macromers can be used for prevention of viral infections and recoveries of biomolecules.